The present invention relates to the depyrogenation of endotoxin-contaminated albumin intended for clinical use. More particularly, the present invention is directed to a method in which a pyrogenic lot of clinical albumin can be made non-pyrogenic again, thus avoiding the monetary loss as well as the loss of precious material.
Pyrogenic albumin is caused by the contamination of endotoxins derived from Gram-negative bacteria during the manufacturing process. Because of the ubiquitous nature of bacteria, the control of these physiologically active agents is of utmost importance to the plasma fractionation industry, as well as to the entire pharmaceutical industry. The most positive method of control, strict aseptic techniques that limit microbial contamination, cannot, in most cases, maintain complete sterility throughout the manufacturing process. Therefore, manufacturers may at times find their final product pyrogenic at the bulk solution stage or after the product has been filled in bottles. The result is the loss of the entire lot of precious material.
In the plasma fractionation industry the monetary loss due to pyrogenic albumin must have been in the millions of dollars per year. However, no plasma fractionator has reported any reliable method to depyrogenate its albumin, except that, in casual conversation, they admitted using charcoal, heat-treatment, or depth filtration, all of which are of "hit or miss" in nature. The only published method for depyrogenation of clinical albumin was reported by Wye and Kim (Vox Sang 32: 182-184, 1977) who mixed pyrogenic albumin with Cohn ethanol Fractions IV-1 and IV-4, based on the findings of Yoshika and Johnson (J. Immunol. 89: 326-335, 1962), who found the endotoxin-inactivating activities in these two fractions followed by differential thermal heating to recover albumin according to the method of Schneider et al. (Blut 30: 121-134, 1975) This reported method not only requires an excessive amount of Cohn Fractions IV-1 and IV-4, but also suffers considerable losses of albumin. The yield based on 21 batches was about 75%. In some cases, the procedure was not able to remove the endotoxins from the pyrogenic albumin solutions, thus resulting in the loss of the entire lot. Moreover, the method using differential thermal heating to recover albumin (Schneider et al, 1975) has not been accepted by the U.S. Food and Drug Administration as a licensed procedure for manufacturing clinical albumin.
It has been known since 1954 (Hegemann, Z. Immunitactsforsch 111: 213-225) that normal human plasma or serum has the ability to diminish the pyrogenicity of endotoxins. This observation was confirmed in subsequent years by many reports (Skarnes et al. J. Exp. Med. 108: 685-700, 1958; Rall et al. Am. J. Physiol. 188: 559-562, 1957; Rudbach and Johnson, Nature 202: 811-812, 1964; Yoshika and Johnson, J. Immunol. 89: 326-335, 1962; Landy et al. J. Exp. Med. 110: 731-750, 1959; Skarnes, Ann. N.Y. Acad. Sci. 133: 644-662, 1966). Yoshika and Johnson (1962) fractionated serum by the Cohn ethanol procedure (Cohn et al. JACS 68: 459-475, 1946) and found that Cohn Fraction IV-1 contains the substance(s) which decreases pyrogenicity caused by endotoxins. These finding were further confirmed by this inventor who isolated from human plasma the protein that is responsible for inactivating such bacterial endotoxins (Hao, Y. L. U.S. Pat. No. 4,677,194, 1987).
The present invention would therefore provide a systematic approach to remove endotoxins from pyrogenic albumin renderig it suitable for clinical use again. The method consists of: (1) titration of a given amount of pyrogenic albumin with increasing amounts of plasma until the mixture gives an endotoxin level equal to or less than 0.5 E.U. (endotoxin units)/ml as assayed by the Limulus Amebocyte Lysate (LAL) tests (Pearson and Weary, Bio Science 30: 461-464, 1980); the minimal amount of plasma required to reach such a low level of endotoxin is the amount of plasma to be added to the pyrogenic albumin for its depyrogenation; (2) mixing the required plasma with pyrogenic albumin in a jacketed tank with cooling, and addition of sufficient saline solution, 0.15M NaCl, to the mixture so that the final protein concentration reaches 1.0-2.0%; (3) adjustment of the pH of the mixture to 5.75.+-.0.05 with 0.8M acetate buffer, pH 4.0, according to a previously reported method of this inventor (Hao, Vox Sang 36: 313-320 1979) and cooling the mixture down to 0.degree. to 1.degree. C.; (4) addition of 95% ethanol to the mixture under stirring until a final ethanol concentration reaches 40-42% (v/v). During the addition of ethanol, the temperature of the mixture should not exceed 0.degree. C. After the required amount of ethanol is added, the mixture should be cooled down to -5.degree. to -6.degree. C. under continuous stirring. The protein concentration of this mixture should be in the range of 0.60-1.2% and the sodium content should be 80-90 milliequivalents, or 0.08 to 0.09M NaCl; (5) stirring the mixture for at least 3 hours at -5.degree. to -6.degree. C., the liquid-solid separation is achieved by centrifugation (Sharples centrifuges AS-16 or AS-26) or by filtration (Hao, Vox sang. 49; 1-8, 1985) at a flow rate of approximately 500 ml/min. The supernatant thus obtained is then filtered through depth filter pads, e.g. Cuno 60S at -5.degree. to 6.degree. C.; (6) precipitation of albumin is carried out by adjusting the pH to 4.8.+-.0.05 with acetate buffer (Hao, 1979). After stirring for at least 3 hours, the liquid-solid separation is again achieved by centrifugation at a flow rate of approximately 500 ml/min or by filtration. The paste (Fraction V) thus obtained can then be considered as the regular Fraction V and processed into non-pyrogenic clinical albumin according to the conventional procedures (Cohn et al., 1946 and Hao, 1979).